Inheritance of Microsatellite DNA Markers in the Pacific Abalone <Emphasis Type="Italic">Haliotis discus hannai</Emphasis>

Microsatellite markers have been developed for a variety of abalones, and locus-specific homozygote excesses at population level have been recorded for microsatellite loci. To ascertain whether null alleles exist at microsatellite loci in the Pacific abalone, we studied the mode of inheritance of 7 microsatellite loci in 4 families with a reciprocal cross of 2 females × 2 males. All loci segregated codominantly, but only 3 loci (Hdh1321, Hdh78, and Hdd108C) conformed to Mendelian segregation and can be used for parental analysis and population genetic studies. When null alleles were considered, 2 loci (Hdh1761 and Hdh1457) confirmed Mendelian expectations in all families, while the remaining 2 loci (Hdd114B and Hdd229) showed deviation from Mendelian segregation in at least one family even though null alleles were considered. These results indicated the need to test the inheritance pattern for microsatellite markers in abalones before using them for population genetic or parentage analysis.     

Population estimators or progeny tests: what is the best method to assess null allele frequencies at SSR loci?

Nuclear SSRs are notorious for having relatively high frequencies of null alleles, i.e. alleles that fail to amplify and are thus recessive and undetected in heterozygotes. In this paper, we compare two kinds of approaches for estimating null allele frequencies at seven nuclear microsatellite markers in three French Fagus sylvatica populations: (1) maximum likelihood methods that compare observed and expected homozygote frequencies in the population under the assumption of Hardy-Weinberg equilibrium and (2) direct null allele frequency estimates from progeny where parent genotypes are known. We show that null allele frequencies are high in F. sylvatica (7.0% on average with the population method, 5.1% with the progeny method), and that estimates are consistent between the two approaches, especially when the number of sampled maternal half-sib progeny arrays is large. With null allele frequencies ranging between 5% and 8% on average across loci, population genetic parameters such as genetic differentiation (F _ST) may be mostly unbiased. However, using markers with such average prevalence of null alleles (up to 15% for some loci) can be seriously misleading in fine scale population studies and parentage analysis.     

Reliability assessment of null allele detection: inconsistencies between and within different methods

Microsatellite loci are widely used in population genetic studies, but the presence of null alleles may lead to biased results. Here, we assessed five methods that indirectly detect null alleles and found large inconsistencies among them. Our analysis was based on 20 microsatellite loci genotyped in a natural population of Microtus oeconomus sampled during 8 years, together with 1200 simulated populations without null alleles, but experiencing bottlenecks of varying duration and intensity, and 120 simulated populations with known null alleles. In the natural population, 29% of positive results were consistent between the methods in pairwise comparisons, and in the simulated data set, this proportion was 14%. The positive results were also inconsistent between different years in the natural population. In the null‐allele‐free simulated data set, the number of false positives increased with increased bottleneck intensity and duration. We also found a low concordance in null allele detection between the original simulated populations and their 20% random subsets. In the populations simulated to include null alleles, between 22% and 42% of true null alleles remained undetected, which highlighted that detection errors are not restricted to false positives. None of the evaluated methods clearly outperformed the others when both false‐positive and false‐negative rates were considered. Accepting only the positive results consistent between at least two methods should considerably reduce the false‐positive rate, but this approach may increase the false‐negative rate. Our study demonstrates the need for novel null allele detection methods that could be reliably applied to natural populations.     

Development of polymorphic microsatellite markers for the zebra finch (Taeniopygia guttata)

To study the population genetics as well as the mating system of captive zebra finch (Taeniopygia guttata) populations, we developed primers for 12 microsatellite loci and screened them in 529 individuals from two successive generations of a single captive population. All markers were polymorphic with five to 14 alleles per locus. We checked all markers for Mendelian inheritance in 307 offspring whose parents were known for sure. Four markers showed evidence for the presence of null alleles. Once allowing for null alleles, we found no mismatches between offspring and parents, suggesting a very low rate of mutation. Average observed and expected heterozygosities across the eight loci showing no evidence for null‐alleles was 0.819 and 0.812, respectively.     

Assessing the genetic diversity in Argopecten nucleus (Bivalvia: Pectinidae), a functional hermaphrodite species with extremely low population density and self‐fertilization: Effect of null alleles

Argopecten nucleus is a functional hermaphroditic pectinid species that exhibits self‐fertilization, whose natural populations have usually very low densities. In the present study, the genetic diversity of a wild population from Neguanje Bay, Santa Marta (Colombia), was estimated using microsatellite markers, and the effect of the presence of null alleles on this estimation was assessed. A total of 8 microsatellite markers were developed, the first described for this species, and their amplification conditions were standardized. They were used to determine the genotype of 48 wild individuals from Naguanje Bay, and 1,010 individuals derived from the offspring of 38 directed crosses. For each locus, the frequencies of the identified alleles, including null alleles, were estimated using the statistical package Micro‐Checker, and the parental genotypes were confirmed using segregation analysis. Three to 8 alleles per locus with frequencies from 0.001 to 0.632 were detected. The frequencies of null alleles ranged from 0.10 to 0.45, with Ho from 0.0 to 0.79, and He from 0.53 to 0.80. All loci were in H‐W disequilibrium. The null allele frequencies values were high, with lower estimations using segregation analysis than estimated using Micro‐Checker. The present results show high levels of population genetic diversity and indicate that null alleles were not the only cause of deviation from H‐W equilibrium in all loci, suggesting that the wild population under study presents signs of inbreeding and Wahlund effect.     

Characterization of microsatellite loci isolated in midget faded rattlesnake (Crotalus viridis concolor)

Primers for five polymorphic microsatellite loci were developed for the midget faded rattlesnake (Crotalus viridis concolor), a rare subspecies of western rattlesnake (Crotalus viridus) found only in parts of Wyoming, Colorado, and Utah. Five polymorphic microsatellites were isolated, four of which had relatively high levels of diversity (eight to nine alleles). We found only two departures from Hardy–Weinberg equilibrium and they occurred in different loci, so null alleles are likely not a problem. Moreover, we found that no two loci were linked. These loci will be applicable for population genetic analysis and perhaps analysis of paternity and mating systems.     

Isolation and characterization of ten microsatellite loci for Senegal sole (Solea senegalensis Kaup)

Senegal sole (Solea senegalensis Kaup) is a high‐value marine flatfish exploited in both fisheries and aquaculture. Here we describe the isolation of seven tetranucleotide and three dinucleotide polymorphic microsatellite loci. Characteristics of a captive broodstock and a wild population are described. Three loci displayed a significant departure from Hardy–Weinberg expectations in both populations, suggesting a substantial frequency of null alleles. The remaining seven loci were found to be in equilibrium in the wild population, whereas only four of them were in the cultured population. Cross‐species amplification was successful for three loci in other five flatfishes.     

Characterization of five microsatellite loci in the Pine Processionary Moth Thaumetopoea pityocampa (Lepidoptera Notodontidae Thaumetopoeinae)

Five microsatellite markers were developed for the lepidopteran species Thaumetopoea pityocampa using an enrichment protocol. All loci could be amplified with no evidence of null alleles and will be useful for population genetic studies. The number of alleles ranged from three to 12 for a population of 30 individuals. Observed heterozygosities ranged from 0.53 to 0.80. No significant heterozygote deficiency was detected. Four markers might be of interest for Th. wilkinsoni.     

Identification of 13 polymorphic microsatellite loci in the zebra finch,Taeniopygia guttata (Passeridae,Aves)

Polymorphic microsatellite loci were identified in order to determine paternity in a captive population of the zebra finch, Taeniopygia guttata. Primer sets from 93 published passerine microsatellite sequences were tested for cross‐species amplification. Thirteen loci were found to be polymorphic, of which, eight displayed null alleles and one locus (Ase50) was found to be Z‐chromosome linked.     

Development of microsatellite loci in pinto abalone (Haliotis kamtschatkana)

Twelve novel di‐, tri‐ and tetranucleotide microsatellite loci to the pinto abalone (Haliotis kamtschatkana) are described. Over 400 individuals were analysed at each microsatellite locus. Observed heterozygosities ranged from 0.44 to 0.93, and numbers of alleles from 20 to 63. Six of the loci contained excesses in homozygosity indicative of inbreeding, nonrandom mating, population admixture, or null alleles.     

Isolation of microsatellites from Girella tricuspidata

Microsatellites were isolated from Girella tricuspidata (Girellidae) and screened for 64 individuals collected from coastal reefs in New South Wales, Australia. All seven loci tested were highly polymorphic, having seven to 42 alleles with average heterozygosities between 0.44 and 1.0. One locus (GT1N8) had a significant excess of homozygotes, probably due to the presence of null alleles. These microsatellite loci should be informative for examining population genetics of this species.     

Microsatellite allele sequencing in population analyses of the South American cactophilic species Drosophila antonietae (Diptera: Drosophilidae)

Drosophila antonietae belongs to the Drosophila buzzatii cluster, a cactophilic group of species naturally endemic to South America. Morphological and genetic analyses indicate that its populations are the most homogenous in the cluster and that the diversity observed is mainly a result of variation within populations. Seven polymorphic microsatellite loci were described for this species and used in the present study to investigate the genetic diversity of natural populations of D. antonietae by both length and sequence variation. The study aimed to understand how homoplasy and null alleles affect inferences about the population history of this species and to obtain an accurate interpretation of population inferences where these loci could be applied. The results provide useful information on the interpretation of genetic data derived from the microsatellite loci described for D. antonietae and on evolutionary aspects of cactophilic Drosophila. Importantly, the results indicate that size homoplasy and null alleles do not represent significant problems for the population genetics analyses because the large amount of variability at microsatellite loci compensate the low frequency of these problems in the populations. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 573–584.     

Isolation and characterization of highly polymorphic microsatellite loci for the garden tiger moth Arctia caja (Lepidoptera: Arctiidae)

A set of microsatellite markers was isolated from Arctia caja, a species that has been declining in UK for several decades. As has been found with other Lepidoptera species, this proved to be a difficult task. Eight enriched libraries identified 103 positive clones, and these yielded only seven polymorphic microsatellites. Allelic diversity ranged from 10 to 23 alleles per locus in a population of 30 individuals. Significant heterozygosity deficits were shown by three of these loci, presumably due to null alleles. The remaining four loci were in Hardy–Weinberg equilibrium and will be used to investigate the population genetic structure of A. caja.     

Microsatellite loci for population and behavioural studies of Horsfield's bronze‐cuckoo (Chalcites basalis: Aves)

Eight polymorphic microsatellite loci were identified for Horsfield's bronze‐cuckoo (Chalcites basalis). These include seven newly isolated loci from cuckoo genomic libraries enriched for GA and GAAA repeat‐containing clones. These loci have a mean expected heterozygosity of 0.71, a mean number of alleles of 13.8 and a combined exclusion probability (one parent known) of 0.9999. Two loci (Cba01 and Cba07) showed a significant deficiency of heterozygotes and may therefore have null alleles, although this effect could be the result of nonrandom population sampling.     

Characterization of 11 microsatellite loci derived from genomic sequences of polychaete Capitella capitata complex

Eleven microsatellite loci derived from the genomic sequence data of Capitella capitata were characterized using 30 samples. The observed number of alleles per locus ranged from four to 36. The levels of observed and expected heterozygosities for polymorphic loci were from 0.10 to 0.87 and from 0.37 to 0.98, averaging 0.52 and 0.77, respectively. Analyses of Hardy–Weinberg equilibrium and genotypic linkage disequilibrium suggest the possible presence of both null alleles and Wahlund effect. One of the 11 loci was difficult to amplify for genotyping. Therefore, the rest 10 loci are good molecular markers for population genetic analysis.     

Isolation and characterization of microsatellite loci from the endangered highlands scrub hypericum (Hypericum cumulicola)

We report the isolation of 19 primer pairs for amplification of polymorphic microsatellite loci for Hypericum cumulicola. These markers were evaluated in 24 individuals from one population; two to four alleles were detected per locus, and observed heterozygosity ranged from 0 to 0.5. Two loci demonstrated significant heterozygote deficiencies, possibly due to null alleles, and significant linkage disequilibrium was found between six pairs of loci. The remaining microsatellite loci will help determine if genetic differentiation is responsible for life‐history differences between natural and anthropogenically disturbed populations of H. cumulicola.     

Polymorphic microsatellite loci from the red urchin,Strongylocentrotus franciscanus,with comments on heterozygote deficit

Strongylocentrotus sea urchins are common subjects for studies in developmental and cell biology, reproductive biology, ecology, and evolution. We report 14 microsatellite loci from the red urchin, S. franciscanus, isolated for the purpose of estimating paternal success of males in experimental group spawns. Most of these loci were found to be highly polymorphic in a population from British Columbia. A high frequency of null alleles appears responsible for heterozygote deficit at a majority of these loci, but if used with appropriate caution, these microsatellites should be effective markers for studies of Strongylocentrotus populations.     

Isolation and characterization of polymorphic microsatellite loci from the pink bollworm Pectinophora gossypiella (Saunders) (Lepidoptera: Gelechiidae)

Thirteen polymorphic microsatellite markers were developed from an enriched partial genomic library for the pink bollworm, Pectinophora gossypiella (Saunders), one of the most important cotton pests in the world. The total number of alleles ranged from two to 12 for a sample of 50 individuals and the expected heterozygosity at these loci ranged from 0.042 to 0.830. Although the presence of null alleles in some loci is suspected, these polymorphic markers are likely to provide useful tools for the population genetic studies of this species.     

Development of 81 new polymorphic EST-derived microsatellite markers for the olive flounder,Paralichthys olivaceus

Expressed sequence tags (ESTs) can be used to identify microsatellite markers. We developed 81 polymorphic microsatellite markers from 4,940 ESTs of the olive flounder, Paralichthys olivaceus. Out of 100 EST-derived microsatellites for which PCR primers were designed, 81 loci were polymorphic in 30 individuals from a single natural population with 2–28 (mean 10.6) alleles per locus. The observed and expected heterozygosities of these loci were 0.033–1.000 and 0.033–0.965, respectively. Segregation analysis within a mapping family revealed non-amplifying null alleles at five loci. These new EST-derived microsatellite markers should be useful for population genetic analyses, pedigree tracing and constructing a linkage map for olive flounder.     

Nine polymorphic microsatellite markers in Crepis sancta (Asteraceae)

We describe nine new variable di‐ and trinucleotide microsatellites in the plant Crepis sancta, as well as conditions for multiplexing and simultaneous genotyping sets of loci. Their variability was assessed in four populations. Four to 33 alleles were detected per locus; two to 23 alleles per population. Significant deviations from Hardy–Weinberg equilibrium could be attributed either to a mating system involving partial selfing or to null alleles. Genetic differentiation over all loci was significant among the four populations studied. These markers will constitute an efficient tool for investigating dispersal patterns in fragmented landscapes. Cross‐species amplification provided few loci variability in three other Crepis species (C. pulcra, C. foetida and C. rubra).